Mathieson was born and raised in Shepparton, Victoria. After leaving school he undertook a hairdressing apprenticeship in the Melbourne suburb of South Yarra. Returning to Shepparton, he operated a salon for fifteen years. Mathieson next moved to the Gold Coast, Queensland, where he operated a hair salon for a further fifteen years.[4]

For much of the 1990s Mathieson and a friend worked in California, exporting vintage Levi's jeans and supplying marble interiors.[5]

Returning to Melbourne in 2004, Mathieson worked for one year at a salon called Heading Out. There he met Gillard, a long-standing client of the salon. They began dating in March 2006 and became partners in 2007.[1] After leaving Heading Out, Mathieson returned to Shepparton and established Tim Mathieson Hair with financial support from his father and brother. However, in 2006 he returned to Melbourne,[6] where he worked as a sales representative for PPS Hairwear, a hair products company.[5] In January 2010, Mathieson was appointed as a property agent for Ubertas Group, a Melbourne real estate agency, and focused on selling high-rise apartments in Melbourne to international buyers.[3] Mathieson would remain in that role until March 2010.[7]

After Julia Gillard became Prime Minister in June 2010, Mathieson supported her by assuming an unpaid, behind-the-scenes role, as well as continuing his involvement with charitable causes.[8]

Mathieson is a divorcé with three adult children.[9] He was previously married to Diane Stark, with whom he had two children.[10] Mathieson also fathered a daughter in his late teens.[11] He started dating Gillard in March 2006 and has remained her partner since.[1] When asked about marriage to Gillard, Mathieson said "We haven't talked about anything more than being spouses at this stage. Wait and see, I guess."[12]

In January 2013, Mathieson attracted media attention for a joke he made while advocating for prostate examinations, advising men to seek out "a small Asian female doctor" when receiving a rectal prostate exam. His remark was considered inappropriate and in poor taste by some commentators. He later apologised "for any offence caused".[16]